Comparative study on in vitro transformation of paralytic shellfish poisoning (PSP) toxins in different shellfish tissues

Dissected tissues of three shellfish species, the Chinese scallop, Chlamys farreri, Manila clam, Ruditapes philippinarurn, and Razor shell, Solen strictu were evaluated for in vitro transformation of paralytic shellfish poisoning （PSP） toxins. Tissue homogenates were incubated with extraction from toxic algae Alexandriurn rninutura to determine toxin conversion. The effects of heating and addition of a natural reductant （glutathione） on toxin conversion were also assessed. The toxin profile was investigated through high performance liquid chromatography with fluorescence detection （HPLC-FLD）. The evident variations in the toxin content were observed only in Chinese scallop viscera homogenates. The concentration of GTX4 was reduced by 45% （approximately 0.8 μmol/dm^3） and 25% （approximately 1 μmol/dm^3） for GTX1, while GTX2 and GTX3 increased by six times （approximately 1 μmol/dm^3） and 3 times （approximately 0.3μmol/dm^3） respectively. Simultaneously, the total toxicity decreased by 38% during the 48 h incubation period, the final toxicity was 20.4 nmol STXeq/g. Furthermore, heated Chinese scallop viscera homogenates samples were compared with non-heated samples. The concentration of the GTX4 and GTX1 was clearly 28% （approximately 0.53 μmol/dm^3） and 17% （approximately 0.69μmol/dm^3） higher in heated samples, GTX2 and GTX3 were four times （0.66 μmol/dm^3） and two times （0.187 μmol/dm^3） lower respectively. GSH （＋） Chinese scallop viscera homogenates samples were compared with GSH （-） samples, the concentration in the GTX4 and GTX1 was 9% （approximately 0.12 μmol/dm^3） and 11% （approximately 0.36 μmol/dm^3） lower respectively, GTX2 and GTX3 was 17% （approximately 0.14 μmol/dm^3） and 19% （approximately 0.006 μmol/dm^3） higher respectively. In contrast,there was a little change in the concentration of PSP toxins of Manila clam and Razor shell tissue ho- mogenates. These observations on three shellfish tissues confirmed that there were species-specific differences in PSP toxins transformation. PSP toxins transformation was more pronounced in viscera tissue than in muscle tissue. PSP toxins was possibly interfered by some carbamoylase enzyme, and the activity in Chinese scallop viscera tissue is more remarkable than in the other two species.

NEOPLASTIC TRANSFORMATION OF HUMAN EMBRYONIC NASOPHARYNGEAL EPITHELIAL CELLS INDUCED BY N,N'-DINITROSOPIPERAZINE (DNP) IN VITRO

This experiment is the first report on N, N'-dini-trosopiperazine (DNF)-induced neoplastic transformation of human embryonic nasopharyngeal (HENPE) cells. The transformed cells showed a prolonged life span, anchorage independent growth, chromosome aberration, tumorigenicity and an altered cell morphological appearance. The results demonstrated that DNP was able to induce not only nasopharyngeal carcinoma (NPC) of rats in vivo, but also neoplastic transformation of HENPE cells in vitro.

Development of High Efficient in vitro Regeneration and Transformation System in Strawberry

In this paper, several factors that affect the efficiency of in vitro adventitious bud regeneration and Agrobacterium tumefaciens-mediated transformation of F. vesca were studied. The results showed that F. vesca seeds germination rate was the highest while seeds were cultured in water, and the germination rate was the lowest while seeds were cultured on MS medium supplemented with hormone; the germination rates that seeds cultured on two and three layers filter paper were higher than that seeds cultured on four and five layers filter paper. In vitro adventitious regeneration efficiency was affected by different explants types. The significant difference was existed between petioles and leaves. When using the same type explants, in vitro adventitious buds regeneration rate and the average number of buds per explant between Ruegen （RE） and Yellow Wonder （YW） had no significant difference. RE to Agrobacterium tumefaciens was more sensitive than YW. Using seedling leaves of RE and YW as materials, an efficient Agrobacterium-mediated transformation system was developed. In this system, the concentration of bacteria was OD600=0.5, the explants were immersed in bacteria broth for 9 min, the co-cultured time was 2 days, and had no pre-cultured time. The percentage of explants with resistant buds of RE and YW was compared. The putative transformed plants were confirmed by PCR.

Efficient Regeneration System for Genetic Transformation of Mulberry (<i>Morus indica</i>L. Cultivar S-36) Using <i>in Vitro</i>Derived Shoot Meristems

Shoot meristems used for the study were exercised from the in vitro regenerated shoots cultured on MS medium supplemented with 0.5 mg/L of BAP for multiplication. The sensitivity of the in vitro regenerated was studied using shoot meristems of 0.5 cm. Shoot meristems were cultured on medium containing 10-100 mg/l kanamycin to determine the concentration that was lethal for multiple shoot induction and root induction. The response of shoot multiplication decreased (66.2%-6.2%) as the concentration of kanamycin increased (10.0-70.0 mg/L) with complete inhibition of shoot proliferation at 100 mg/L kanamycin. The rooting phase was very sensitive to kanamycin compared to shoot multiplication. The percentage of shoots that rooted decreased (53.8%-4.8%) with increase in the concentration of kanamycin (10.0-70.0 mg/l) on IBA and 2,4-D supplemented medium. For transformation studies, the shoot tips that were infected with Agrobacterium strain were placed on selection medium containing MS medium with 0.5 mg/L BAP and 100 mg/L kanamycin and scored for the putative transformed shoots. An average of 62.2% of shoot tips developed shoot buds from the base and the shoots reached a length of 0.5-1.0 cm at the end of 30 days of culture on the selective medium in comparison to control which showed no response. An average of 66.7% of the regenerated plants showed GUS expression on selection medium where 43.2% and 65% of GUS expression was recorded in the leaves and callus. Leaves and callus induced from the controls did not show GUS activity. Stable integration of nptII gene with the genomic DNA from these transformed plants was confirmed through PCR analysis. Our result presents an efficient regeneration system using in vitro derived shoot meristems for Agrobacterium mediated gene transfer.

In Vitro transformation of LW13 rat liver epithelial cells

A rat liver epithelial cell line designated LW13 was established using a sequential sedimentation method. The cell line retained many normal properties of liver epithelial cells and showed some structural and functional features resembling those of liver parenchymal cells. LW13 cells became malignant after the introduction of exogenous transforming EJ Ha ras gene. Tumors produced by inoculation of the transformed cells into baby rats .contained areas of poorly differentiated hepatocellular carcinoma. In situ hybridization analysis confirmed the random rather than specific integration of exogenous ras gene into host chromosomes. Furthermore , an at least tenfold increase in the expression of the endogenous c myc gene was detected among transformed cell lines, suggesting the involvement of the c myc proto oncogene in the in vitro transformation of rat liver epithelial cells by EJ Ha ras oncogene.

Agrobacterium tumefaciens-mediated transformation of Eucalyptus urophylla clone BRS07-01

Genetic transformation is becoming routine for engineering specific traits in important clones of recalcitrant species such as Eucalyptus;however,the efficiency is still low for most species,so many researchers still use seeds instead of clones as initial explants.This work aimed to develop a genetic transformation protocol,based on a highly efficient in vitro organogenesis protocol,for an Eucalyptus urophylla clone selected in our breeding program.Plant growth regulators were evaluated for indirect organogenesis and rooting.In a two-step protocol,the combination of callus induction media supplemented with 0.5 μM thidiazuron+0.5 μM naphthaleneacetic acid(NAA)and shoot induction media supplemented with 5.0 μM benzylaminopurine+1.0 lM NAA allowed up to 85.6%shoot formation with more shoots per explants when compared with other concentrations.Transgenic plants expressing the uidA gene were obtained using Agrobacterium tumefaciens and selected for kanamycin resistance.A RAPD analysis was used to check for somaclonal variation.In tests using 11 RAPD primers,we did not observe somaclonal variation in the in vitro stages evaluated.

Hepatitis C virus in human B lymphocytes transformed by Epstein-Barr virus in vitro by in situ reverse transcriptase-polymerase chain reaction

AIM: To study persistence and replication of hepatitis C virus (HCV) in patients' peripheral blood mononuclear cells (PBMC) cultured in vitro. METHODS: Epstein Barr virus (EBV) was used to transform the hepatitis C virus from a HCV positive patient to permanent lymphoblastoid cell lines (LCL). Positive and negative HCV RNA strands of the cultured cells and growth media were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) each month. Core and NS5 proteins of HCV were further tested using immunohistochemical SP method and in situ RT-PCR. RESULTS: HCV RNA positive strands were consistently detected the cultured cells for one year. The negative-strand RNA in LCL cells and the positive-strand RNA in supernatants were observed intermittently. Immunohistochemical results medicated expression of HCV NS3 and C proteins in LCL cytoplasm mostly. The positive signal of PCR product was dark blue and mainly localized to the LCL cytoplasm. The RT-PCR signal was eliminated by overnight RNase digestion but not DNase digestion. CONCLUSION: HCV may exist and remain functional in a cultured cell line for a long period.

ELECTRON MICROSCOPIC OBSERVATION OF SIMIAN SARCOMA VIRUS TRANSFORMED NIH 3T3 CELLS IN VITRO

For electronic microscopic observation, we found SSV-transformed NIH 3T3 cells were different from non-transformed cells. In SSV-transformed NIH 3T3 cells nuclei cytoplasma ratio was increased and in cytoplasma the ribosomes (polyribosomes were attached to the swollen rough endoplasmic reticulum. It was likely that ribosomes were lined together functionally and structionally to produce specific protein (PDGF-like protein).

The effect of endogenous transforming growth factor β_1 on the growth of bladder cancer cells in vitro

Objective To investigate the influence of endogenous transforming growth factor β1(TGFβ1) on the cell cycle regulation and proliferation of bladder cancer. Methods A constructed replication defective retroviral vector pRevTβ-AS, which carried antisense RNA of TGFβ1.was transfected to a bladder cancer cell line EJ. The proliferation and clone-formation of transferred cells were observed in vitro,and the alteration of cell cycle was also detected by flow cytometric analysis. Results TGFβ1 antisense RNA was transferred into EJ cell and expressed efficiently. After the inhibition of target gene expression in EJ cells, the reduced growth and clone-formation rates were demonstrated, and the proliferative indexes were decreased by 12 % . The ratios of GO and G1 stage cells to June 2003 Vol12 No2 the antisense RNA-transfected EJ cells were increased, simultaneously,the ratio of S stage cells to the antisense RNA-transfected EJ cells ratios were decreased, compared with the control group. Conclusion The

In vitro regeneration of Populus tomentosa from petioles

A reliable in vitro regeneration procedure for Populus tomentosa is a prerequisite for its trait improvement through genetic transformation. We established a systematic protocol for indirect regeneration of P. tomentosa using in vitro petioles of Chinese poplar cultivar ＇fasta-3＇. A high frequency of callus induction （〉97 %） was obtained from isolated petioles cultured on the modified 1/2MS basal medium supplemented with 0.5 mg/L ZT and 1.0 mg/L NAA, and the tested calli were subsequently plated on 1/2MS basal medium supplemented with 0.25 mg/L BA, 0.25 mg/L ZT, 0.25 mg/L NAA, 0.01 mg/L TDZ, and 0.5 mg/L KT for efficient regeneration of shoots after being cultured for 6 weeks. The regenerated shoots were vigorously rooted on the tested media supplemented with 1.0 mg/ L IBA and 0.5 mg/L NAA. These results can facilitate genetic transformation of P. tomentosa for trait improvements in future.

胚胎培养液中表皮生长因子、转化生长因子、血管内皮生长因子与胚胎发育、妊娠结局的关系

目的分析胚胎培养液中表皮生长因子(EGF)、转化生长因子(TGF)、血管内皮生长因子(VEGF)与胚胎发育及体外受精-胚胎移植(IVF-ET)后妊娠结局的关系。方法选取2020年2月至2021年6月在唐山市妇幼保健院就诊并接受IVF-ET的92例病人为研究对象,根据妊娠结局将其分为妊娠组54例和非妊娠组38例;采用Wetzels胚胎评分系统进行胚胎评级,根据评分将胚胎分为Ⅰ级组、Ⅱ级组、Ⅲ级+Ⅳ级组。留取胚胎培养液,采用酶联免疫吸附(ELISA)法检测胚胎培养液中EGF、TGF-β1、VEGF表达水平;采用受试者操作特征(ROC)曲线分析胚胎培养液EGF、TGF-β1、VEGF水平对妊娠结局的预测价值;采用logistic回归分析影响IVF-ET后妊娠结局的因素。结果Ⅲ级+Ⅳ级组、Ⅱ级组、Ⅰ级组EGF[(0.46±0.19)、(1.14±0.21)、(2.68±0.81)μg/L]、TGF-β1[(58.72±15.13)、(80.68±19.12)、(98.74±23.75)ng/L]表达水平依次升高,VEGF[(39.66±12.71)、(22.85±4.79)、(13.86±3.32)ng/L]表达水平依次降低(P<0.05)。与妊娠组[(2.54±0.73)μg/L、(85.44±19.87)ng/L、(15.94±3.61)ng/L]相比,非妊娠组EGF[(1.49±0.43)μg/L]、TGF-β1[(60.42±15.76)ng/L]表达水平较低,VEGF[(24.75±7.94)ng/L]表达水平较高(P<0.05)。EGF、TGF-β1、VEGF水平及三者联合预测妊娠结局的曲线下面积(AUC)分别为0.88[95%CI:(0.78,0.97)]、0.88[95%CI:(0.78,0.98)]、0.81[95%CI:(0.68,0.95)]、0.96[95%CI:(0.90,1.00)],特异度分别为82.4%、82.4%、94.1%、88.2%,灵敏度分别为83.3%、94.4%、66.7%、96.4%。VEGF是IVF-ET后妊娠失败的独立危险因素(P<0.05),TGF-β1是IVF-ET后妊娠失败的保护因素(P<0.05)。结论胚胎培养液中EGF、TGF-β1、VEGF表达水平可能对评估胚胎质量及预测妊娠结局有重要价值。

离体培养条件下莱氏绿僵菌致病形态和非致病形态的转录组分析

莱氏绿僵菌[Metarhizium rileyi(Farlow)Kepler]是一种重要的昆虫病原真菌,在寄生过程中有致病和非致病两种形态,但只有致病形态能杀死寄主。为了解莱氏绿僵菌由非致病形态向致病形态转变的分子机制,本研究对这两种形态的菌体进行转录组分析。结果表明,两种形态的菌体共得到89498个Unigenes,GO注释中与“细胞组分”有关的基因占比最高;KOG注释中与“主要功能预测”有关的基因最多;KEGG注释的“代谢”子类中与“代谢通路”有关的基因最多,在“环境信息过程”子类中与“MAPK信号途径”有关的基因最多,在“细胞过程”子类中与“群体感应”有关的基因也有较高的表达,这初步为莱氏绿僵菌二型性转变的群体感应调节机制提供了基因证据。差异分析表明,莱氏绿僵菌形态转变过程中有3113个基因表达上调,3671个基因表达下调,但差异富集分析未得到有效的结果。综上,莱氏绿僵菌形态转变时基因表达有明显的变化,代谢旺盛、触发群体感应机制并激活细胞信号转导途径,最终完成非致病形态向致病形态的转变。本研究可为深入了解莱氏绿僵菌致病的分子机制提供有益的借鉴。

间充质干细胞外泌体对体外肝星状细胞增殖和活化的影响

目的探讨间充质干细胞来源的外泌体(MSCs-Exo)对人肝星状细胞系LX-2细胞增殖及活化的影响.方法取MSCs-Exo,在透射电子显微镜下观察.取LX-2细胞,分为对照组、5μg/L、10μg/L和20μg/L转化生长因子-β1(TGF-β1)处理组,采用RT-PCR和Western blot法检测细胞α-SMA和COL1A1 mRNA及其蛋白表达.取TGF-β1诱导活化的LX-2细胞,以50μg/mL、100μg/mL和200μg/mL MSCs-Exo干预24 h,采用MTT法检测细胞增殖.结果经电镜观察及粒径分析表明,实验所用MSCs-Exo符合外泌体的形态特征;实验所用MSCs-Exo阳性标志蛋白CD9和CD63阳性,而GM130蛋白阴性;经TGF-β1诱导活化的LX-2细胞α-SMA和COL1A1 mRNA水平及其蛋白表达均显著升高;当TGF-β1诱导浓度为20μg/L时,细胞α-SMA和COL1A1 mRNA及其蛋白表达量最高;与对照组比,MSCs-Exo-100组和MSCs-Exo-200处理细胞增殖活力显著降低(均P<0.0001);50μg/mL、100μg/mL和200μg/mL MSCs-Exo作用TGF-β1诱导活化的LX-2细胞α-SMA mRNA水平和COL1A1 mRNA水平较对照组显著降低(P<0.01),细胞α-SMA和COL1A1蛋白水平也较对照组显著降低(P<0.0001);MSCs-Exo-100和MSCs-Exo-200处理的LX-2细胞增殖活力较对照细胞显著降低(均P<0.0001).结论MSCs-Exo可以抑制TGF-β1诱导的LX-2细胞增殖和活化,其应用研究值得期待.

柳树离体再生及遗传转化体系研究进展

柳树因其药用价值、生态修复能力及作为生物能源的潜力而受到关注。近些年已培育出许多优良品种,并在柳属植物基因组中定位到与经济性状及抗逆性相关的候选基因,建立离体再生及遗传转化体系可以获得大量优质柳树苗,并对候选基因进行功能研究。为了更清晰地认识影响柳树再生及遗传转化的不同因素,本文对柳树离体再生和遗传转化的研究进展进行了总结,主要包括柳属不同种初代培养、继代增殖培养、生根培养的最佳培养基的筛选,诱导愈伤组织最佳的培养基、激素、外植体类型的筛选,遗传转化时最佳的农杆菌的类型、浓度、侵染时间、预培养和共培养时间及选择压等因素的筛选。目前柳树的愈伤组织再生和遗传转化仍存在难点,本研究对其未来进一步深入研究方向进行了展望,以期对柳树的离体再生及遗传转化体系建立提供参考,为后续柳树基因工程研究及柳树的遗传改良奠定基础。

PHENOLIC COMPOUNDS PROMOTE HIGH EFFICIENCY TRANSFORMATION OF PLANT EXPLANTS in vitro BY Agrobacterium tumefaciens

The development of plant gene engineering requires a simple, rapid and efficient system for genetic transformation. It is recently demonstrated that certain compounds, secreted by host plant cells, are capable of eliciting the activation of Agrobacterium and Ti plasmid genes which are involved in promoting T-DNA transfer from the bacterium to the plant cell,

石榴离体再生体系建立及遗传转化技术研究进展

石榴果实营养丰富,树形优美,花色鲜艳,果形奇特,是一种兼具食用观赏价值的经济树种,在国内深受大众喜爱。我国石榴种质资源丰富,栽培历史悠久。然而近年来由于栽培技术落后,生产管理粗放,导致品种退化、树势减退。随着生活水平提高和经济的发展,人们对石榴的品质要求日益提高,因而培育优质果苗,筛选优良品种成为石榴生产的必然趋势。采用离体培养技术及构建遗传转化体系,对培育脱毒苗和选育优良品种提供了技术手段。通过对国内外研究成果进行收集分析,对石榴离体培养及遗传转化技术、现状、问题分析及未来研究发展趋势等进行阐述,以期为开展石榴的科学研究和生产实践提供理论基础和科学依据。

核桃体细胞胚发生与转基因研究进展

总结了核桃体细胞胚发生的研究进展 ,列表统计已报道的核桃 5个种和 3个杂种体细胞胚发生的外植体与诱导条件 ,重点论述了影响核桃体细胞胚发生与次生胚发生的因素 ,介绍了核桃体细胞胚萌发与转化的方法。还总结核桃转基因研究的进展 。

茶树生物技术研究进展

总结了近40a来茶树生物技术的研究进展,包括器官培养、细胞与组织培养、原生质体培养与细胞融合、遗传转化,及其在茶树快速繁殖、遗传改良、种质保存、次生物质生产等方面的应用,并初步分析了茶树生物技术的发展趋势.

转化人胚肌腱细胞的体外培养及生物学特性研究

目的研究经ｐｔｓＡ５８Ｈ质粒转化的人胚肌腱细胞的生物学特性，探讨细胞生长特性与体外培养条件改变的关系。方法取人胚肌腱细胞和经ｐｔｓＡ５８Ｈ质粒转化的人胚肌腱细胞，进行体外培养。对细胞进行形态学、超微结构及一般特征的观察。在不同培养条件下进行生长曲线绘制，平板克隆实验和软琼脂培养以及免疫组织化学法胶原染色。结果在正常培养条件下，人胚肌腱细胞和转化人胚肌腱细胞的生长性状几乎无差异，且冻存复苏并不改变其生长特性。在３９．５℃及３５℃进行条件培养，转化细胞的生长特性发生改变。转化细胞能长期传代，增殖能力强，并存在接触抑制现象和血清营养依赖性，在软琼脂中不能生长，具有分泌Ⅰ型胶原的能力。结论经ｐｔｓＡ５８Ｈ质粒转化的人胚肌腱细胞可长期连续传代。通过改变培养条件可控制其生长。无肿瘤细胞的生物学特点。

我国葫芦科植物离体培养研究进展

葫芦科植物包括多种瓜类蔬菜,对其进行离体培养研究具有重要的理论和实践意义。综述了国内在葫芦科植物器官培养、体细胞胚胎发生、花药培养、原生质体培养和体细胞杂交及离体遗传转化等方面取得的研究进展,并对葫芦科植物离体培养、遗传转化与育种的前景作了展望。